Creation of huge libraries of organic compounds, usable for e.g. drug screening, is a convenient and economical means for developing new pharmaceuticals. Two out of several ways of creating such libraries are traditional solid support synthesis and display technologies taking advantage of nucleic acid directed synthesis. Solid support synthesis has been the traditional means for creating huge libraries of compounds, such as peptides, poly-carbohydrates and small molecule organic compounds. Nucleic acid directed synthesis, on the other hand, provides a more efficient means for effectuating reactions by utilizing the willingness of complementary nucleic acid strands to hybridize and thus bringing the reactants into proximity to each other, thereby enhancing the rate of reaction.
Display technologies have been developed to combine information storage and amplification capabilities of nucleic acids with the functional activities of other compounds. These display technologies rely on an association between a functional entity and a nucleic acid sequence which is informative about the structure of the functional entity.
An advantage of such methods is that very large libraries can be constructed and probed for a desired activity of the functional entities. Library members having the desired activity can then be partitioned from library members not having the desired activity, thus creating an enriched library with a higher fraction of members having the desired activity. This process is called selection. The structures of the library members in the enriched library can then be identified by their cognate nucleic acid sequence, thus allowing identification even from minute amounts of material.
Nucleic acid directed synthesis suffers from some limitations, namely that the nucleic acids are incapable of hybridizing in predominantly organic solvents and that hybridization is not error free occasionally resulting in incorrect matching of strands.
The first mentioned limitation means that the nature of the reactions is limited to water compatible reactions. This excludes reactions involving reactants that are insoluble in water, i.e. the reactants will not be able to “meet” the compound attached to the nucleic acids and therefore the rate of reaction will be heavily compromised; and reactions that are inhibited by water or proceed through a water incompatible mechanism.
The other limitation has an impact when creating large libraries. Firstly, when improper matching occurs the duplex may not be stable enough in order for the reaction to occur. Furthermore, it is necessary to employ systems, which can be selected and amplified with a maximum accuracy in order to enrich and identify the potential drug candidates.
Rozenman and Liu (Mary M. Rozenman and Davis R. Liu; DNA-Templated Synthesis in Organic Solvents; ChemBioChem 2006 7, 253-256) describe a method for performing DNA-templated Synthesis (DTS) in organic solvents using short (10-30 bp) DNA-duplexes of various conformations, i.e. i) in a simple end-of-helix architecture with juxtaposed reactants, ii) in a long-distance end-of-helix architecture with ten intervening nucleotides between the hybridized reactants, iii) in the “omega” architecture, described in e.g. WO04/016767A2 below, with a five-base loop (Ω5) and iv) with reactants linked to non-complementary (mismatched) oligonucleotides. Rozenman and Liu show that such short structures are capable of remaining hybridized when transferred from an aqueous solvent into an organic solvent (up to 99.9% (V/V)) without the use of quaternary ammonium salts. This finding is contrary to previous studies where it was believed that the presence of such salts, which associate with the DNA phosphates in order to stabilize the duplex, was necessary for retention of the DNA-duplex in organic solvents (See e.g. K. Ijiro, Y. Okahata, J. Chem. Soc. Chem. Commun. 1992, 18, 1339-1341 and K. Tanaka, Y. Okahata, J. Am. Chem. Soc. 1996, 118, 10679-10683).
Using this method Rozenman and Liu have performed, in an organic solvent, various chemical reactions, which either require reagents that are insoluble in water or proceed through water incompatible mechanisms. Rozenman and Liu show that proper matching of the oligonucleotides into duplexes is a prerequisite for the reactions to proceed. It is therefore of utmost importance to a successful reaction that a nucleic acid system is employed, which provides a high degree of proper matching.
Thus, it has been shown that it is indeed possible to perform DNA templated synthesis in an organic solvent with short DNA duplexes when using the method of Rozenman and Liu. This method could be implemented in library construction technologies.
Similarly, in vitro display technologies taking advantage of the flexibility of organic chemistry and rounds of selections, amplification and diversification have been described. These methods rely on the use of templates.
One example is described in WO00/23458, using a “split and mix” principle a development of the conventional solid support synthesis. A library of ssDNA templates is used, each containing a chemical reaction site and several positions of codon segments. The templates are compartmentalized by virtue of hybridizing to a repertoire of anti-codon sequences for a given codon position. Then, a compartment specific chemical reaction is performed modifying the reaction site on the templates. The templates are then mixed and the process reiterated by using other codon positions to form a combinatorial library.
Another example is described in WO02/074929A2, using a “single-pot” principle. A library of oligonucleotide templates is used, each containing a chemical reaction site and several positions of codon segments. Furthermore, using a repertoire of transfer units, the method consists of an oligonucleotide anti-codon sequence and a chemical reactive group. The library of templates are hybridized with a repertoire of transfer units for a given codon position. This brings the chemical group on a hybridized transfer unit in proximity to the reaction site on the hybridized template, which consequently guides the chemical reaction of cognate pairs. This process is then reiterated using other codon positions to form a combinatorial library.
A limitation of the above proximity guiding of cognate pairs of code and chemical group is given by the linear structure of the template oligonucleotide. As a consequence of the linearity the distance between codon and the chemical reaction site will differ from codon position to codon position. For codon positions longer away from the reaction site the proximity guiding becomes compromised, as the local concentration drops to the power of three as a function of the distance. This disadvantage becomes more pronounced for complex libraries, with more codon positions and more complex codons.
This problem is sought solved in WO04/016767A2, where the transfer units apart from an anti-codon segment also contain a constant segment, which is complementary to a constant sequence on the template close to the reactive site. Thus, by hybridizing a transfer unit to a template, the template sequence between the codon position in question and the constant segment is bulging out, to form a so-called omega structure. The concept is that the codon segment is responsible for the specificity and the constant segment responsible for the proximity. Also suggested in WO04/016767A2 is a so-called T-architecture of the templates, where the reactive site on the template is situated in the middle of the template, with the codon positions spread out on each side. Consequently, the distance problem is so called “cut in half”.
WO 2004/056994A2 discloses a method similar to WO02/074929A2 or WO04/016767A2 with the difference that the template is cut into minor sequences, termed “connecting polynucleotides” in the application. The connecting polynucleotides connect transfer units to bring these into reaction proximity. In certain embodiments the connecting polynucleotides may comprise a reactive chemical group. To obtain an encoded molecule the method is dependent upon codon/anti-codon recognition prior to reaction.
The template directed libraries described above are subsequently subjected to selections to form enriched libraries. The enriched libraries members' synthetic history can then be deduced through the encoding nucleic acid. The enriched libraries can also be amplified and diversified by for example error prone PCR, thus allowing for molecular evolution.
A limitation in these approaches is that a large number of templates have to be created, which is cumbersome, as the templates have to be of considerable length to ensure proper codon/anti-codon hybridization. In methods using a plurality of minor sequences to make up the final directed synthesis, the number of sequences to be synthesized is even higher than the actual library size.
The prior art methods using templates suffer from the disadvantage that the encoding is dependent upon hybridization of codon and anti-codon sequences. Sometimes hybridization between single stranded oligonucleotides will happen without perfect complementarities. In the case of library construction the result is loss of the association between the encoding and the synthetic history. Consequently, upon selection, positive codes may be de-selected and negative codes may be selected. For more complex libraries this disadvantage becomes more significant as the complexity of the single stranded oligonucleotides also increases, both with respect to numbers, length and sequences. This makes the processes more difficult to control
As described above, in vitro display technologies allowing display of a variety of compound classes, selections, amplifications and diversifications have been developed. However, there is still an ongoing need for improvement, especially with respect to the quality in library construction and of diversification. The present invention offers a method for producing an encoded molecule in which the method does not require the pre-synthesis of a large number of templates or templates at all. Furthermore, the present method is not dependent upon codon/anti-codon recognition for an encoded molecule to be formed. Finally the present invention takes advantage of the establishment of a super nucleic acid structure prior to reaction, which allows for reaction of the chemical groups in a solvent predominantly consisting of an organic solvent.